Hypoxia is an important factor mediating tumor progression and therapeutic resistance, in part through proteome changes mediated by the transcription factor hypoxia-inducible factor (HIF)-1. Since glioblastoma multiforme is the epitome of a highly aggressive tumor entity, while lower-grade astrocytomas often show a prolonged clinical course, a profound difference in the extent of hypoxic tissue areas and corresponding magnitude of HIF-1 activity may exist between these entities. In this study, to address this question, serial sections of 11 glioblastomas and 10 anaplastic astrocytomas were immunostained for HIF-1α, glucose transporter (GLUT)-1, carbonic anhydrase (CA) IX (i.e., hypoxia-related markers), Ki67 (proliferation), phosphorylated ribosomal protein S6 [p-rpS6; mammalian target of rapamycin (mTOR) activity] and CD34 (microvascular endothelium). Digital scans of whole tumor sections were registered to achieve geometric correspondence for subsequent morphometric operations. HIF-1α-, GLUT-1- and CA IX-positive staining was found in all 11 glioblastomas, showing a preferential expression in tissue areas adjacent to necroses. A considerable spatial overlap between GLUT-1 and CA IX, and a colocalization of these proteins with areas of enlarged mean diffusion distances were observed. Conversely, 8 of the 10 anaplastic astrocytomas were completely negative for hypoxia-related markers. The glioblastomas also showed significantly greater heterogeneity of intercapillary distances, larger diffusion-limited tissue fractions, significantly higher mTOR activity and a trend for higher proliferation rates. Microregionally, mTOR and proliferation showed a significant spatial overlap with areas of shorter mean diffusion distances. In conclusion, diffusion-limited hypoxia, leading to the expression of hypoxia-related markers is a pivotal element of the glioblastoma phenotype and may be driven by dysregulated growth and proliferation in normoxic subregions.